Project Summary Obstructive sleep apnea (OSA) is a debilitating condition leaving 14%-49% of middle-aged adults with excessive daytime sleepiness and impaired cognitive, metabolic, and cardiovascular functioning. Treatments beyond the often poorly tolerated CPAP remain limited, despite this unmet medical need. This Program Project seeks to identify new intervention points in neural brain circuits to combat OSA. In OSA, the activity of muscles that keep the airway open drops leading to airway collapse and impeded ventilation. Tissue and blood oxygen levels fall and carbon dioxide (CO2) levels rise (hypercapnia). This leads to an intensification of breathing movements, meant to improve ventilation, but which only worsen the airway collapse by generating negative pressure. Once a threshold of hypercapnia is reached, the brain arouses, rapidly leading to robust airway opening, resumption of ventilation, and return to sleep. This OSA cycle occurs many times per night, fragmenting sleep and inflicting autonomic surges underlying vasoconstriction and tachycardia that drive much of the comorbidity. Our Program seeks to identify neural brain circuits to optimize the activity of muscles that keep the airway open and to augment breathing (Respiratory Augmentation) without arousing the brain. In Project 4, we focus on the serotonergic neuronal system in the lower brainstem in mice, leveraging our team?s recent discovery of two genetically distinct subtypes of serotonergic neurons each critical for a robust ventilatory response to hypercapnia, yet each acting on largely distinct components of the neural respiratory arousal circuit. The subtype denoted Egr2-Pet1 increases respiratory drive by connections to centers that measure pCO2 and that mediate arousal, whereas the second subtype, denoted Tac1-Pet1, sends connections to motor centers that control airway dilation and inspiration. By modulating these subtypes separately it may be possible to a) optimize airway dilator tone to avoid airway closure, b) optimize ventilation to avoid exacerbation of airway closure, c) optimize EEG arousal threshold to reduce sleep fragmentation, and d) avoid cardiovascular stress. In Aim 1, we will study how Egr2-Pet1 and Tac1-Pet1 populations modulate each of these different functions during wake, sleep, and arousal, enabled via intersectional chemogenetic tools in combination with a Repeated-CO2-Arousal model for OSA, while measuring diaphragm and airway dilator muscle activity, brain activity, heart rate, and respiratory rate and depth. In Aim 2, we will use in vivo and in vitro optogenetic and calcium imaging tools to determine circuit nodes to which Egr2-Pet1 and Tac1-Pet1 neurons functionally connect, relevant for airway dilation, ventilation, and arousal. In Aim 3, we will visualize the circuit nodes from which Tac1-Pet1 and Egr2-Pet1 neurons receive input. Throughout, we will query response dependence on serotonin receptors, relevant for pharmacological strategies in OSA. Collectively, results from this work have the potential to transform our understanding of and approaches to prevent OSA.